Molecular phenotypes of notochordal cells purified from immature nucleus pulposus

Abstract

The immature nucleus pulposus (NP) is populated by cells of notochordal-origin that are larger and contain an extensive cytoskeletal network and numerous vacuoles. The disappearance of these cells with age is believed important in regulating metabolic shifts that may contribute to age-related disc degeneration. The precise biological function of these notochordal cells in the immature NP remains unclear, however, because of challenges in studying the mixed cell population in the NP. In this study, notochordallike cells were purified from immature NP cells using a new fluorescence-activated cell sorting (FACS) protocol with auto-fluorescence and size analysis. The unique molecular phenotypes of sorted notochordallike cells were characterized by the mRNA expression pattern for key matrix proteins and modulators, and by the expression of cell-matrix receptor integrin subunits. An FACS analysis showed that the immature NP contained a majority of cells that were larger than anulus fibrosus (AF) cells and with fluorescence higher than AF cells. In comparison with the small NP cells separated by the FACS protocol, sorted notochordal-like cells expressed lower mRNA levels of type I collagen, biglycan, TIMP1, HSP70 and c-fos, and did not express detectable mRNA levels of decorin, lumican, multiple MMPs or IL-1b via realtime quantitative RT-PCR. A greater number of these notochordal-like cells also expressed the higher levels of a6, a1 and b1 integrin subunits as compared to small NP cells. Together, our results point towards a unique molecular phenotype for these notochordal-like cells of NP, characterized by the absence of gene expression for specific small proteoglycans and higher protein expression of integrin subunits that regulate interactions with collagens and laminin. Future studies will be important for revealing if this unique molecular profile is coordinated with functional differences in pericellular matrix regions and/or integrin-mediated cell-matrix interactions for these notochordallike cells within the NP.

The evolutionary importance of cell ratio between notochordal and nucleus pulposus cells: an experimental 3-D co-culture study

“…Furthermore, non-invasive femtosecond laser microscopy revealed clear size differences between these two cell types and seems to point toward two different cell lineages, if cell shape and size are considered [15]. Furthermore, the NC differs by the presence of large vacuoles, which can be separated by the size-scatter of FACS analysis [5]. These large vacuoles found in NC of the intervertebral disc has been attributed a possible functional role in osmoregulation [18].…”

“…With ageing, these presumably progenitor-like cells disappear in some species and in other species they persist up to adulthood [5,28]. In human, they disappear early in childhood [19].…”

An understanding of intervertebral disc development, maturation and cell phenotype provides clues to direct cell-based tissue regeneration therapies for disc degeneration

“…For this to be possible, a comprehensive phenotypic characterization of notochordal cells, and especially of markers specific to the notochordal cells in the human IVD would be required. To date, several attempts have been made to identify notochordal markers, either by comparing the gene expression of immature notochordal rat NP with its costal cartilage [73], rat NP tissue with different degrees of maturity [74][75][76], comparing immature pig NP with AF cells [74,77], sorted large and granular with smaller, less granular pig NP cells [78], immature human NP with AF cells [74] or NP from human juvenile scoliotic and adult discs [79] (Tables 1, 2, 3, 4 list putative notochordal/ immature NP markers identified in different species and describe their relevance to the IVD field). Proposed rat notochordal markers identified in these studies were CD55 [73], brachyury, neuropilin (Nrp-1), CD221, BASP-1 [76], N-Cad [73,76], TGF-b, BMP-6 and CTGF [75].…”

Tissue engineering and the intervertebral disc: the challenges

“…Research into directing stem cells or notochordal cells towards a discogenic phenotype is hindered by lack of specific markers which can distinguish nucleus pulposus (or even annulus fibrosus or CEP) cells from articular chondrocytes or nucleus cells from annulus cells [23,58]. Markers commonly used for showing conversion of stem cells to a disc cell phenotype are expression of aggrecan and type II collagen and of SOX-9 [91]; these are no different, however, from those used to identify articular chondrocytes.…”